CORE--HEMATOPOIESIS LABORATORY

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A central goal of several projects proposed by the Manhattan Comprehensive Sickle Center is to modulate the gamma- and/or beta globin synthesis program of hematopoietic cells from patients in order to ameliorate the symptoms of sickle cell disease. The specific aims of Dr. Weinberg's Hematopoiesis Core Laboratory are: 1) To provide tissue culture methods and support to component projects. Tissue culture methods and techniques which are established and used extensively in the core lab include: a) methylcellulose cultures for evaluation of hematopoietic progenitor cells (HPC), b) short term ex vivo cultures for retroviral mediated gene transduction and expansion of stem-progenitor cells (S/PC), c) long term cultures for retroviral mediated gene transduction of S/PC, expansion of S/PC, and assay of long term cultures initiating cells (LTC-IC), and 2) To perform globin chain synthesis studies in transduced cells derived from cultures, mice transplanted with transduced cells, and transgenic animals using polyacrylamide gel electrophoresis and fluorography. Projects, experiments, and core's role are as follows: 1) Project PI: G. Atweh. Experiments: Transduction and expansion of HPC, analysis of colonies derived from transduced cells, globin synthesis by transduced cells and colonies. Role of core: short term liquid transduction cultures, two phase liquid cultures, methylcellulose cultures and gel electrophoresis and fluorography. 2) Project PI: A. Bank. Experiments: Transduction and expansion of HPC. Analysis of transduction in colonies derived from long term culture- initiating cells (LTC-IC) and HPC, analysis of globin synthesis by cells and colonies. Role of core: Long term marrow cultures +/-stroma, +/- hematopoietic growth factors (HGFs), short term transduction cultures +/- stroma, gel electrophoresis and fluorography. 3) Project PI: F. Constantini:Experiment: Analysis of globin chain synthesis in transgenic mice.colonies. Role of core: Reticulocyte incubations, gel electrophoresis, fluorography. 4) Project PI: J.Bieker,Experiments: Culture HPC with EKLF-like zinc finger proteins, analyze globin synthesis in cells and colonies from these cultures. Role of Core: Methylcellulose and two phase liquid cultures, gel electrophoresis and fluorography 5) Project PI: S. Piomelli. Experiments: HPC cultures +/-arginine butyrate, pre-, during, and post- in vivo arginine butyrate, examine globin synthesis by colonies by colonies. Role of core: Methylcellulose and gel electrophoresis and fluorography.